Selenium is an essential micronutrient in the diet of humans and other mammals. It has many known health benefits such as preventing various forms of cancer (e.g., colon, prostate, lung and liver), preventing heart disease and other cardiovascular and muscle disorders, serving as an antiviral agent, and playing roles in delaying the aging process, delaying the progression of AIDS in HIV positive patients, in immune function, mammalian development and male reproduction. Even though many human clinical trails have been undertaken to elucidate the role of this element in cancer prevention, delaying the progression of AIDS, etc., at the cost of hundreds of millions of dollars, little is known about the mechanism of how selenium acts at the molecular level in exerting these many health benefits. We proposed that the health benefits are due largely to the presence of this element in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focuses on 1) developing mouse models to assess the role of selenium and specific selenoproteins in human health and cancer prevention and 2) the means by which Sec is biosynthesized and incorporated into protein. The project discussed herein examines our research on the role of selenium in health and cancer prevention. During the past year, we finished and published our project on knocking down and knocking in selenoprotein expression providing an alternative and novel means of studying protein function. One of the selenoproteins we used as a model in developing this technique was thioredoxin reductase 1 (TR1). Since TR1 is overexpressed in many cancer cells, and since this selenium-containing oxidoreductase has been proposed to be a target for cancer therapy, we knocked down its expression in a mouse lung cancer cell line (LLC1) and reported that most of the malignant phenotypes were reversed more towards those of normal cells. These studies have been expanded to show that the malignant phenotypes of numerous human and mouse cell lines can also be altered by TR1 knockdown yielding a similar cancer inhibitory effect. We obtained an oncogenic, Ras overexpressing cell line from NIH3T3 cells and knocked down TR1 to analyze the underlying mechanism of the resulting growth inhibitory properties and found that the cells had a defect in the S phase. We are currently examining the precise mechanism for this inhibition. In addition, during the last few years, we have developed several transgenic, knockout, conditional knockout and knockdown cell and mouse lines that perturb Sec tRNA synthesis which in turn modulate selenoprotein expression as models to better understand selenium and selenoprotein metabolism and their roles in health. Using loxP-Cre technology, we have targeted the removal of selenoprotein expression in several tissues and organs. One of our major focuses in this area has recently been on the role of selenoproteins in immune function and skin cell function. We targeted the removal of selenoprotein expression in T-cells and in macrophages to investigate immune function and in mouse epidermis to investigate skin cell function. We previously found that T cells deficient in selenoproteins exhibit defective T cell proliferation, phosphorylation of ERK1/2, IL2R&#945; expression and an increased rate of apoptosis. This past year we completed in vivo immunization studies, measurement of ROS production and rescuing the defective proliferation. In vivo challenging studies showed that T cell selenoprotein deficient mice are immuno-compromised as the knockout mice exhibit significantly lower levels of various Igs upon challenging compared to sibling, wild type controls. The defective T cells were also found to generate and accumulate higher amounts of ROS upon stimulation; and upon using an external source of antioxidant, defective T cell proliferation was completely rescued. Removal of selenoproteins in macrophages showed an accumulation of ROS in unstimulated selenoprotein KO macrophages compared to wild type controls. Cytokine analysis of bone marrow-derived macrophages following in vitro stimulation with lipopolysaccharide (LPS) showed a decrease in IL-6 and TNF-&#945; production while in vivo serum cytokine analysis appeared to show a significant increase in TNF-&#945; production following injection with LPS. However, an in vivo challenge with LPS showed no significant difference in LPS-induced lethal shock between KO and control mice. An examination of the LPS-induced pro-inflammatory signaling cascade is currently being investigated. In our skin selenoproteinless mice, the animals exhibited stunted growth, which became more prominent with age, and they have a much shorter life span as compared to normal littermates. The knockout mice have flakiness and multiple wrinkles in skin, and pathological analysis revealed moderate epidermal hyperplasia along with acute coagulative necrosis of the epidermis and oral mucosa. We recently initiated a liver cancer study to examine the role of selenoproteins in liver cancer prevention. This study was undertaken as an extension of our previous finding that mice survive with the complete removal of selenoproteins in their liver and in response to the request by both houses of the US Congress that the National Cancer Institute should devote more effort to liver cancer research as the increased incidence of liver cancer and the small number of effective treatments is in sharp contrast to many other forms of cancer where the incidence is declining and the treatment options are rapidly increasing. These studies are in the early stages and we are examining the response of the selnoproteinless mice to carcinogens